1. Field of Invention
This invention relates to a novel process for the production of cyclic alternan tetrasaccharide from alternanase using oligosaccharides as substrates.
2. Description of the Prior Art
Alternan, an extracellular D-glucan produced by Leuconostoc mesenteroides (Jeanes et al., 1954, J. Am. Chem. Soc., 76:5041-5052), and its lower molecular weight hydrolysis products have been previously described. Early studies of the alternan found that the compound was considerably resistant to microbial degradation and was also not attacked by enzymes that degrade starch, nigeran or pullulan (Cote, 1992, Carbohydrate Polymers, 19:249-252). The only enzymes that were reported to hydrolyze alternan to any significant extent were isomaltodextranases and alternanase. Of these, isomaltodextranases were not endo-hydrolases but rather exo-hydrolases or exo-dextranases. Two isomaltodextranases were examined for hydrolysis of alternan (referred to as B-1335 fraction S), the isomaltodextranases produced by Arthrobacter globiformis (Sawai et al., 1978, Carbohydrate Res., 66:195-205) and by an actinomycete Actinomadura (Sawai et al., 1981, Carbohydrate Res., 89:289-299). The authors concluded that the isomaltodextranases release mainly isomaltose units from the non-reducing ends of alternan chains that are terminated with an xcex1-1,6-linked D-glucopyranosyl residues. Later, studies with A. globiformis isomaltodextranase purified in this laboratory according to Okada et al. (1988, J. Biol. Chem., 5:495-501) confirmed that this enzyme also was not capable of endo-hydrolytic cleavage of alternan, but functioned in an exo-fashion.
Alternanase was described more recently by Cote et al. as an endo-xcex1-D-glucanase specific for alternan, having substantially greater activity toward alternan than dextran (U.S. Pat. Nos. 5,786,196, 5,888,776, and 5,889,179). This enzyme is produced and secreted extracellularly by a plurality of soil bacteria. Among the fractions present in the thinned alternan resulting from hydrolysis with alternanase are a previously unknown cyclic tetrasaccharide, cyclo{-6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1-} and derivatives thereof. This cyclic tetrasaccharide may be used as a metal salt complexing agent, a soluble, low- or non-caloric substitute for sucrose, and as bulking agents or extenders in foods and cosmetics.
I have now discovered that the cyclic tetrasaccharide, cyclo{-6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1-}, may be produced by alternanase hydrolysis of complex carbohydrates other than alternan. Surprisingly, panose, pullulan, xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-D-Glc, and D-glucans having alternating xcex1-(1,6) and xcex1-(1,4) linkages, are not only hydrolyzed by alternanase, but the hydrolysis of these complex carbohydrates also produces the cyclic tetrasaccharide cyclo{-6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1-}. In this process, the cyclic tetrasaccharide is produced by contacting a solution of one or more of the above-mentioned complex carbohydrates with an amount of alternanase under conditions effective for activity of the enzyme. Furthermore, the substrate panose used in the reaction may be produced from a variety of polysaccharides or oligosaccharides, including starch, maltose, pullulan, and mixtures thereof.
In accordance with this discovery, it is an object of this invention to provide an improved, novel process for producing the cyclic tetrasaccharide cyclo{-6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1-}.
Another object of this invention is to provide a process for the production of the cyclic tetrasaccharide from common, readily available polysaccharides or oligosaccharides, particularly starch.
Other objects and advantages of this invention will become obvious from the ensuing description.
The process of the invention produces the cyclic tetrasaccharide, cyclo{-6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-xcex1-D-Glcp-(1-}, which has the following structure (I): 
This structure is intended to show linkage only, and no particular conformation is implied. As noted above, this cyclic tetrasaccharide, as well as the enzyme alternanase, were described in the previous patents of Cote et al. (U.S. Pat. Nos. 5,786,196, 5,888,776, and 5,889,179, the contents of each of which are incorporated by reference herein). In the processes disclosed in those prior patents, the cyclic tetrasaccharide was formed by hydrolysis of alternan, a polysaccharide of alternating glucose units linked in an alternating xcex1-1,3- and xcex1-1,6-fashion, with alternanase.
In contrast with the previously described process, I have surprisingly found that not only will alternanase hydrolyze polysaccharides other than alternan, but it will hydrolyze selected complex carbohydrates to produce the same cyclic tetrasaccharide. Starting complex carbohydrates suitable for use herein as substrates for production of the cyclic tetrasaccharide include panose, pullulan, xcex1-D-Glcp-(1,6)-xcex1-D-Glcp-(1,3)-D-Glc, D-glucans having alternating xcex1-(1,6) and xcex1-(1,4) linkages, and mixtures thereof, with use of panose being preferred. However, although pullulan may be used, reaction rates and yields of the cyclic tetrasaccharide are significantly lower than those obtained using panose. The activity of alternanase toward these substrates is particularly unexpected as these polysaccharides do not contain the alternating xcex1(1,3),xcex1(1,6)-D-glucosidic linkage sequences previously thought to be necessary for alternanase activity.
In accordance with the process of this invention, a catalytically effective amount of alternanase may be contacted with one or more of the above-mentioned selected polysaccharide or oligosaccharide substrates in an aqueous solution under conditions effective to hydrolyze the polysaccharide. Alternanase generally retains hydrolytic activity over pH and temperature ranges between about 4.5 to 9 and about 0xc2x0 to at least 50xc2x0 C., respectively, with optima at about pH 7 and about 40xc2x0 C. for the enzyme from strain NRRL B-21195. At a pH of 7.0, enzyme activity decreases rapidly as the temperature is increased to 60xc2x0 C. The presence of calcium ions in the reaction mixture is required for optimal activity. Addition of the calcium binding agent EDTA has been found to inhibit activity.
Yields of cyclic tetrasaccharide will vary considerably with the particular substrate utilized, with optimal yields being obtained from use of panose. The extent of reaction may be controlled by terminating the reaction at any time. A variety of techniques which are conventional in the art may be used to stop the reaction, including heating to denature the enzyme, addition of inhibitors (e.g. EDTA), or adjusting the pH.
Following completion of the reaction, the cyclic tetrasaccharide produced may used in crude form, although it is preferably recovered from the reaction mixture in pure or substantially pure form. The particular technique for separation is not critical, and a variety of techniques are suitable for use herein. With the exception of the cyclic tetrasaccharide, the reaction products are all reducing sugars and thus may be removed using conventional ion-exchange resins. In the preferred embodiment, the cyclic tetrasaccharide may be selectively isolated from the other components of the reaction mixture by a single pass through a basic ion-exchange resin, which will bind the reducing sugars such as panose and maltose, but will allow the non-reducing cyclic tetrasaccharide to pass therethrough. Strong anionic exchange resins are particularly preferred for use herein, including, for example, AMBERLITE IRA-400 (Rohm and Haas, Philadelphia, Pa.) and DOWEX AG 1X (Dow Chemical, Midland, Mich.). Alternatively, the cyclic tetrasaccharide may be separated from the reaction mixture by chromatography, such as silica gel chromatography.
As starting materials in the reaction of the invention, the above-mentioned complex carbohydrates may be provided in substantially pure form or, in the alternative, they may be provided as a mixture or in impure form. Furthermore, although alternan does not interfere with the reaction and may be present, the reaction is preferably conducted substantially in the its absence.
In a preferred embodiment, the substrate panose is produced from reaction of a polysaccharide or oligosaccharide, such as starch, maltose, maltodextrins, pullulan, and mixtures thereof, whereupon it may be subsequently converted to the cyclic tetrasaccharide by action of alternanase. The particular process for the production of the panose is not critical, and a variety of techniques have been described which are suitable for use herein. Without being limited thereto, in one particularly preferred embodiment, maltose may be converted to panose by transglucosidation with known glucosyltransferase or glucosidase/transglucosidase preparations. For example, Hang and Woodams (1995, Biotech. Letters, 17:1335-1336, and 1997, Letters in Applied Microbiology, 24:43-46, the contents of each of which are incorporated by reference herein) disclosed that the glucosyl transferases of Aureobasidium species or Aspergillus foetidus (crude enzyme preparations or culture filtrate) or commercially available cytolase preparations all effectively converted maltose to panose. In another particularly preferred embodiment, starch or a maltodextrin or a mixture of maltodextrins may be hydrolyzed to maltose by action of xcex1-amylase or xcex2-amylase (alone or in combination with an xcex1-[1,6]-glucosidase) such as described by Lehninger (Biochemistry, Second Edition, Worth Publishing, New York, 1975, pp. 264-265, the contents of which are incorporated by reference herein), which maltose may then be converted to panose as described above. Although pullulan may be directly hydrolyzed to the cyclic tetrasaccharide as disclosed hereinabove, reaction rates and yields are low. However, rates and yields may be increased significantly by hydrolyzing pullulan to panose by action of neopullulanase as described by Kuriki et al. (1992, J. Fermentation and Bioengineer., 73:198-202, the contents of which are also incorporated by reference herein), which panose is then converted to the cyclic tetrasaccharide by alternanase.
Panose for use as a substrate herein may also be produced from simpler saccharides, such as by reversion of D-glucose. In one embodiment, treatment of concentrated glucose syrups with acid or with xcex1-glucosidase or glucoamylase will generate a mixture of di- and oligosaccharides, including panose.
Panose produced from a polysaccharide or oligosaccharide or from a simple saccharide in any of these techniques, may be converted to the cyclic tetrasaccharide by alternanase as described hereinabove. Moreover, the reaction of panose to cyclic tetrasaccharide may be conducted concurrently with the reaction(s) for generation of panose, or the reactions may be conducted separately. The skilled practitioner will recognize however, that when the reactions are conducted concurrently yields may be relatively lower, particularly if the conditions for optimal activity of the various enzymes used are different.
Production of alternanase utilized in this invention was described in Cote et al. (U.S. Pat. Nos. 5,786,196, 5,888,776, and 5,889,179) as mentioned hereinabove. In review, alternanase was originally isolated from soil bacteria that were selected for the ability to produce extracellular enzymes which hydrolyzed alternan in an endo-fashion, and seven strains of soil bacteria identified as belonging to the genus Bacillus were isolated which produced and secreted extracellular alternanase. All seven strains have been deposited under the Budapest Treaty in the United States Department of Agriculture, Agricultural Research Service culture collection in Peoria, Ill., and have been assigned deposit numbers NRRL B-21189, B-21190, B-21191, B-21192, B-21192, B-21193, B-21194 and B-21195. Of these, strain NRRL B-21195 produces the highest level of alternanase activity and is preferred. Alternanase produced from any of these strains will hydrolyze the polysaccharide substrates in the same manner to produce the cyclic tetrasaccharide.
Alternanase production may be accomplished by culture of any of the aforementioned bacterial strains, isolates or subcultures having the identifying characteristics of those strains, mutants of those strains capable of producing alternanase, or other isolates recovered by the screening procedure described hereinbelow, by conventional techniques under aerobic conditions that are effective to promote growth and alternanase production. Any number of well-known liquid or solid culture media may be used, although growth on liquid media is preferred as the enzyme is secreted into the media and recovery is simplified. Without being limited thereto, particularly preferred culture media include Brain-Heart Infusion Broth or Trypticase Soy Broth. Similarly, the media may contain a variety of carbon sources which will support growth and production of the enzyme, including but not limited to glucose, starch, maltose and alternan. The presence of alternan in the culture medium is not essential for production of the enzyme, although optimal alternanase production is achieved by addition of alternan thereto. The precise degree of enhancement is variable and is dependent upon the particular strain used. For example, production of the enzyme is greatly increased by addition of alternan when using strains NRRL B-21189, B-21190, B-21191, B-21192, B-21192, B-21193 and B-21194. In contrast, strain B-21195 is a constitutive producer of alternanase, and addition of alternan effects a less dramatic increase in alternanase production, approximately between 20 to 30%. The amount of alternan added to the media is not critical and may be readily determined by the practitioner skilled in the art. The bacteria will grow and produce alternanase over wide pH and temperature ranges, with a pH of about 7.0 and a temperature of about 30xc2x0 C. being preferred.
Upon completion of the fermentation, typically between 24 to 96 hours, alternanase may be isolated or separated from the microorganisms using techniques conventional in the art, such as by centrifugation or filtration. As a practical matter, it is envisioned that commercial formulations of alternanase may be prepared directly from liquid culture medium from which cells have been removed in this manner, thereby obviating the need to further purify the enzyme.
Optionally, the alternanase remaining in the culture medium may be further concentrated and purified, particularly for applications demanding a high degree of purity where contamination by other enzymes, microbial products, monosaccharides, or culture media components may be undesirable. Suitable techniques for concentration and/or purification of alternanase may be readily determined by the practitioner skilled in the art and include, for example, dialysis, ion-exchange chromatography, and preferably HPLC size-exclusion chromatography and electrophoresis, particularly polyacrylamide-gel-electrophoresis (PAGE). Using these techniques, alternanase may be recovered in pure or substantially pure form. It is also envisioned that the enzyme may be formulated in conjunction with a suitable inert carrier or vehicle as known in the art. The skilled practitioner will recognize that such carriers must be compatible with the enzyme. Without being limited thereto, details of the preferred fermentation and separation procedures are described in Example 3 and in Biely et al. (1994, Eur. J. Biochem., 226:633-639, the contents of which are incorporated by reference herein).
For commercial applications, the cyclic tetrasaccharide may be used directly in crude form in the reaction mixture or, in the alternative, separated from the reaction mixture as described above. In either event, the cyclic tetrasaccharide may be used in a variety of applications, particularly as bulking agents or extenders in foods and cosmetics, as soluble, low- or non-caloric sucrose substitutes, or as metal salt complexing agents.
The cyclic tetrasaccharide may optionally be further modified, with such derivatives being useful in binding and complexing metal salts. Without being limited thereto, preferred derivatives include O-alkyl ethers, O-acyl esters, partially or fully sulfated esters, and particularly ionic carboxymethyl and diethylaminoethyl derivatives. These derivatives may be prepared at one or more sites on the cyclic tetrasaccharides using techniques conventional in the art, such as described by Yalpani (1988, Polysaccharides: Syntheses, Modifications and Structure/Property Relations, Elsevier Press, New York, pgs. 234-299) or Kennedy and White (Bioactive Carbohydrates: In Chemistry, Biochemistry and Biology, Halsted Press, New York, pgs. 63-65), the contents of each of which are incorporated by reference herein.